Antibodies to human gastrin-releasing peptide precursor and use thereof

ABSTRACT

Antibodies made to a flanking region GPR(31-8) in human gastrin-releasing peptide (GRP) precursor. Since the antibodies have high affinity to GRP precursor, and the GPR precursor is highly stable in the blood, then lung cancer, especially small cell lung cancer can be diagnosed with high reliability by detecting or measuring GRP precursor in the blood of a patient using the present antibodies.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antibodies to human gastrin-releasingpeptide (GRP) precursor, and the use of the antibodies as a diagnosticagent for cancer.

2. Related Art

It is known that cancer cells produce substances specific to the cancercells as well as substances common between normal cells and cancercells. It has become possible to diagnose the characteristics of cancercells and patients with cancer by measuring the cancer-specificsubstance. Substances specifically produced by cancer cells includeoncogene products and growth factors, which are responsible foroncogenesis, growth and developments of cells. Moreover, it isconsidered that the production of carcinoembryotic proteins, hormonesand enzymes and the like are characteristics of the oncogenesis.Therefore, if one of substances which define cancer cells, i.e.,so-called tumor markers can be assayed with high sensitivity, diagnosisof cancers becomes possible.

Since the presence of neuroendocrine particles in small cell lung cancerwas observed in 1968, it has been asserted that small cell lung canceris derived from neuroendocrine cells, and at present, is included inAPUD (amine precursor uptake and decarboxylation)-type tumors, and isdistinguished from other non-small cell lung cancers which areepithelial tumors. It has been known that the small cell lung cancershows the characteristics of the neuroendocrine cells in acytobiological study, and is reported to produce peptide hormones suchas serotonin, adrenocorticotropic hormone (ACTH), calcitonin, gastrinreleasing peptide (GRP) etc., to exhibit high L-dopa decarboxylase(L-DDC) activity characteristic to APUD-type cells or tumors, and toexhibit high activities of neuron specific enolase (NSE) and creatinekinase BB (CK-BB), which are specific to neuron cells.

Analysis of a surface antigen of small cell lung cancer based on variousbiological properties of the cells as indicators using a monoclonalantibody was started with a report by Minna, Science 214, 1246-1248,1981, and has been well developed by a lot of researchers. Assay systemsso far practically used as tumor markers include those usingcarcinoembryonic antigens such as carcinoembryonic antigen (CEA),α-fetoprotein (AFP), Carbohydrate Antigen 125 (CA125); enzymes such asNSE, L-DDC, CK-BB; hormone-related substances such as ACHT, alcoholdehydrogenase (ADH). However, the positive ratio for sera of patientswith cancer obtained using the above assay systems is at most 50 to 60%,while frequently patients having cancer provide negative result.

So far, GRP is known as one of tumor markers of small cell lung cancer.GRP is a peptide consisting of 27 amino acids extracted from the stomachof porcine by McDonald in 1978, and has activities to stimulatesecretion of gastric acid, various hormones etc. There are threeprecursor proteins different in their C-terminal structure due toalternative splicing of RNA. Recently, the production of GRP as anautocrine growth factor in small cell lung cancer was found, and it isinterested as a tumor marker.

In patients with small cell lung cancer, about 80% of the cases provideincreased blood GRP concentration. Moreover, even in the cases of earlyphase, the blood GRP concentration is in an increased level, andtherefore it is promised that a diagnostic of cancer using GRP as amarker is highly effective. However, conventional assay methods for GRPuse antibodies to an active peptide, GRP(1-27), and its sensitivity istoo low to be practically used. It is supposed that one of main reasonsof difficulty to measure a serum GRP concentration using an antibody toGRP(1-27) is instability of GRP(1-27) in the blood.

Holst et al. (J. Clin. Oncol. 7, 1831-1838, (1989)) developed aradioimmunoassay (RIA) system using polyclonal antibodies to a syntheticpeptide corresponding to the portion from 42 to 53 positions ofC-terminal flanking peptide of GRP precursor, and demonstrated that GRPprecursor protein can be a powerful diagnostic marker for small celllung cancer. However said system was not practical because it providedan insufficient positive ratio due to its sensitivity. Because theantigen protein used was a part of GRP precursor, resulting antibody hadlow sensitivity and specificity body. Moreover, the low sensitivity ofthe assay system needed an extraction of the analyte protein from alarge amount of a sample, resulting in difficulty in clinicalapplication of the system. GRP precursor protein present in the blood isa macromolecule having a molecular weight of 8,000 to 100,000. Therefor,in an assay system using antibodies to GRP(42-53) which is a part of GRPprecursor, sensitivity and specificity are limited.

In normal cells, a protein is produced in the rough-surfaced endoplasmicreticulum, concentrated in the Golgi apparatus resulting in formation ofsecretory granules in which the protein is packed, and extracellularlysecreted through so-called regulated pathway. Since the secretorygranules contain proteolytic enzymes, a precursor of the protein can beadequately processed during passing through the regulated pathway. Onthe other hand, in cancer cells, since the rough-surfaced endoplasmicreticulum is remarkably developed while the number of the secretorygranules is small, then when GRP is produced and secreted, GRP precursoris extracellularly secreted through the constitutive pathway from therough-surfaced endoplasmic reticulum without being affected by action ofany proteolytic enzymes, rather than passing through the regulatedpathway involving the secretory particles. Therefore, the blood ofpatients with cancer contains precursor GRP and flanking peptides inaddition to an active peptide GRP (1-27).

Compared with GRP(1-27), the active site-free flanking peptide of GRPprecursor is expected to be stably present at a high concentration inthe blood.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides novel antibodies to a regionso far not used as an antigen in a GRP precursor and capable ofdiagnosis of lung cancers with a high sensitivity, and a lung cancerdiagnostic agent comprising the antibody.

In accordance with the present invention, novel antibodies are providedusing a peptide having an amino acid sequence from the 31st Ser to the98th Asp of GRP precursors as an antigen. The peptide contains commonsequence among 3 GRP precursors but not a GRP active site in GRPprecursors. The antibodies have a high affinity specificity to GRPprecursors and therefore are useful for diagnosis of lung cancers.

More specifically, the present invention provides an antibody to apeptide having the amino acid sequence shown in SEQ. ID NO: 1, andreactive with human GRP precursors.

The present invention further provides a monoclonal antibody to humanGRP precursor having a dissociation constant Kd of 3×10⁻⁸ ˜5×10⁻⁷ M forimmunocomplex formed from the monoclonal antibody and the human GRPprecursor.

The present invention further provides a lung cancer diagnostic agentcomprising said antibody and a carrier.

The present invention still more provides hybridomas producing saidmonoclonal antibody.

The present invention further provides a process for production of ahuman GRP precursor antigen, comprising culturing a host transformedwith an expression vector comprising a coding region coding for theamino acid sequence shown in SEQ ID NO: 1, and recovering the human GRPprecursor antigen from the culture. Note, "proGRP" means precursor ofGRP.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 represents a nucleotide sequence of DNA coding for proGRP(31-98)and of oligonucleotides for synthesizing the DNA.

FIG. 2 represents a process for the construction of an expressionplasmid pAT-TrpE-proGRP(31-98) for the production of proGRP(31-98).

FIG. 2 represents an example of measurement of GRP precursor in serafrom patients with small cell lung cancer, using anti-GRR (31-98)antibodies of the present invention.

FIGS. 4A and 4B are graphs proGRP showing that (31-98) is more stablethan GRP 1-27in serum or plasma. FIG. 4A shows the stability of GRP1-27and FIG. 4B shows the stability of GRP(31-98).

FIG. 5 is a graph showing affinity to proGRP of various monoclonalantibodies of the present invention.

FIG. 6 represents an example of calibration curve for ELISA using thepresent monoclonal antibodies 3G2 and 2B10 in a mixture as firstantibodies and rabbit anti-proGRP polyclonal antibody as secondantibody.

PREFERRED EMBODIMENT FOR CARRYING-OUT THE INVENTION (1) Antigen proteinand process for preparation thereof

In a process for in vivo production of GRP, three GRP mRNAs aregenerated by alternative splicing of a mRNA, and three precursorproteins having a common amino acid sequence up to the 98 positionincluding the active site but having different C-terminal structures areformed. The GRP precursor antigen used in the present invention is apeptide having an amino acid sequence from the 31th Ser to 98th Asp ofthe precursor protein, which is common between three GRP precursors andprotein, which is common between three GRP precursors and does notcontain GRP active region. The number of amino acids used in the presentinvention is determined by taking the N-terminal amino acid val ofmature GRP as the first position. Therefore, the mature GRP isrepresented by GRP(1-27), and the present antigen peptide is representedby proGRP(31-98).

An amino acid sequence of the present GRP precursor antigenproGRP(31-98) and an example of nucleotide sequences coding therefor areshown in SEQ ID NO: 1.

The antigen peptide of the present invention can be prepared by chemicalsynthesis or genetic engineering. Since the chemical synthesis ofpeptides having more than 40 amino acids is difficult, geneticengineering is preferable.

The production of the present antigen peptide by genetic engineering canbe carried out by transforming a host such as E. coli with an expressionvector capable of expressing the present antigen peptide in the hostsuch as E. coli, culturing the transformant, and recovering the antigenpeptide from the cultured bacterial cells.

Although the above-mentioned expression vector may contain anynucleotide sequence coding for the present antigen polypeptide, in thecase of an E. coli host, a nucleotide sequence comprising codonsfrequently used in E. coli and not containing palindrome is preferable.

As a nucleotide sequence coding for the amino acid sequence ofproGRP(31-98), the nucleotide sequence shown in SEQ ID NO: 1 ispreferable.

A promoter such as a tryptophan promoter is present upstream of thecoding region for the present peptide to enhance transcriptionefficiency in a host used such as E. coli. Although the peptide codingregion may be present immediately downstream of the promoter, the codingregion may be present downstream of a coding region for a proteininherently produced by a host such as E. coli Trp E protein, asdescribed hereinafter. In the latter case, the present peptide may beobtained as a fusion protein.

An expression vector of the present invention, as with conventionalexpression vectors, contains a selective marker such as antibioticresistance and an origin of replication for replicating in a host suchas E. coli. Moreover, a translational stop codon is positioneddownstream of the peptide coding region. As a starting material for theconstruction of the expression vector, for example, pUC9, pBR322, andother vector such as commercially available vectors may be used.

The above-mentioned expression vector may be constructed by synthesizingthe present peptide coding region by a known synthetic process such asphosphoramidite method, and cloning it into a vector such as a knownvector expressed in a host such as E. coli host. In Examples describedhereinafter, the GRP coding region was inserted into TrpE gene or TGF˜αgene (see Japanese Patent Application No. 63-28908) present downstreamof E. coli tryptophan promoter.

Transformation with an expression vector can be carried out according toa conventional procedure. Culturing conditions of host such as E. coliis also conventional.

The present peptide produced by a host such as E. coli transformed witha vector can be isolated and purified by, for example, collecting cellsby, for example, centrifugation, disrupting the cells by well knownlysozyme treatment and/or ultrasonication, and applying the disruptantto a gel filtration chromatography or the like. Definite conditions forisolation and purification of the peptide is described hereinafter inExamples.

(2) Production of antibody

Mouse such as Balb/C mouse is periodically immunized by peritoneal orsubcutaneous administration of the present antigen alone or as anantigen prepared by binding the present antigen with a hapten such asbovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) etc., in amixture with Freund's complete adjuvant. After antibody titer in theblood is increased, the mouse is boosted by administrating the presentantibody through a tail venous of mouse. After that, the spleen isremoved and spleen cells are fused with appropriate myeloid cells suchas mouse myeloid cells. This procedure can be carried out according toKohler and Milstein, Nature 256, p495-497, 1975, to obtain hybridomas.

The hybridomas thus obtained are cultured in an appropriate medium, anda hybridoma cell line producing an antibody specifically reactive withthe present antigen is selected and cloned. Next, monoclonal antibodyproduced is recovered by, for example, column chromatography.

Polyclonal antibodies can be prepared by periodically immunizing ananimal such as a guinea-pig, rabbit, goat, sheep, etc., with the presentantigen alone, or in a form of conjugate with bovine serum albumin (BSA)or keyhole limpet hemocyanin (KLH) etc. by means of a foot pad,intramuscular injection or subcutaneous injection, as a mixture withFreund's complete adjuvant. An increase of antibody titer in the bloodis tested, the whole blood is obtained and polyclonal antibody isrecovered by column chromatography or the like.

Monoclonal antibody or polyclonal antibody thus obtained can be used tocarry out ELISA or other immunoassays such as IRMA, RIA, FIA, CIA, etc.to measure GRP precursor for example in a blood sample.

EXAMPLES

The present invention will now be further illustrated by, but notlimited to, the following examples.

Example 1

Preparation of proGRP(31-98)

(1) Construction of cloning vector

As shown in FIG. 1, GRP gene is divided into DNA fragments of about 60bases, and each DNA fragment was synthesized by phosphoramidits method.

    ______________________________________                                        Fragment  SEQ ID NO   Fragment  SEQ ID NO                                     ______________________________________                                        H1        3           L1        7                                             H2        4           L2        8                                             H3        5           L3        9                                             H4        6           L4        10                                            ______________________________________                                    

The synthesized DNA fragments were purified by reversed phasechromatography and ligated by T4 DNA ligase to obtain the GRP gene.

The gene thus obtained having a restriction enzyme EcoRI site and SalIsite at the 5'- and 3'-termini respectively was inserted into anEcoRI/SalI-digested cloning vector pUC9. The ligation product was usedto transform E. coli JM107, which was then cultured overnight in Lmedium in the presence of 40 μg/ml ampicillin, isopropyl thiogalactoside(IPTG) and X-gel to obtain a candidate strain.

Plasmid was extracted from the candidate strain, and the nucleotidesequence of the inserted gene was tested by Sanger method to confirmthat the inserted gene has an expected nucleotide sequence. E. colicontaining a cloning vector comprising a desired GRP gene was designatedas pUC-proGRP(31-98)/JM107, and the cloning vector was designated aspUC-proGRP(31-98).

(2) Construction of expression vector (FIG. 2)

The vector pUC-proGRP(31-98) was digested with EcoRI and SalI, and a GRPgene fragment of about 220 base pairs was extracted. On the other hand,an expression vector pAT-TrpE-TGF˜α was digested with restrictionenzymes EcoRI and SalI to obtain a larger fragment, which was thenligated with the GPR gene fragment using T4 DNA ligase. The ligationproduct was used to transform E. coli HB101, which was then culturedovernight in L medium in the presence of 40 μg/ml ampicillin to obtain acandidate strain. The candidate strain was designated aspAT-TrpE-DROGRP(31-98)/HB101, and the expresstion vector thus obtainedwas designated as pAT-TrpE-DROGRP(31-98).

(3) Purification of expressed polypeptide

The above-constructed strain containing the expression vector,pAT-TrpE-DROGRP(31-98)/HB101 was cultured and the expression ofrecombinant protein was tested. Namely, the strainpAT-TrpE-DROGRP(31-98)/HB101 was cultured overnight in 32 ml of LBmedium containing 40 μg/ml ampicillin, and the resulting culture wasinoculated to 3.2L of M9 medium containing 0.5% casamino acid andculturing was carried out at 37° C. When the absorbance at 600 nmreached 0.4, indolacrylic acid (IAA) was added to the culture to a finalconcentration of 30 μg/ml, and culturing was further continued for 20hours. The culture was centrifuged to collect 10 g of cells. Thecollected cells were suspended in a 100 mM Tris-HCl (pH 8.0) buffercontaining 2 mg/ml lysozyme and 2 mM EDTA, and the suspension wasallowed to stand at 0° C. for 30 minutes. The suspension was furtherultrasonicated and centrifuged to obtain an insoluble fraction as aprecipitate.

The insoluble fraction thus obtained was solubilized by the addition of8M urea, and centrifuged to collect a supernatant. The supernatant wassubjected to a column chromatography using DEAE TOYOPEAL (TOSO Co.)(eluate: A=20 mM Tris-HCl/6M urea (pH 8.0); B=0.5M NaCl/eluate A (pH8.0); concentration gradient: from A to B with linear gradient/300minutes; column φ1.6×40 cm; flow rate 1 ml/min.) to isolate and purifythe desired protein. The eluted fractions were gathered, dialysed andlyophilized. The protein was cleaved with cyanogen bromide to removeTrpE moiety. The cyanogen bromide cleavage was carried out by adding theprotein to a 70% formic acid solution to a protein concentration of 1%,adding 100 equivalent amount of cyanogen bromide thereon, and theresulting reaction mixture was allowed to stand at 37° C. for 24 hours.The mixture was dialysed, lyophilized and subjected to reversed phasehigh performance liquid chromatography (eluate: A=0.1% trifluoroaceticacid/water, B=60% acetonitryle/0.1% trifluoroacetic acid/water) topurify and obtain 30 mg of GRP protein. Purity of the protein washomogeneous as determined by reversed phase column chromatography andSDS-polyacrylamide gel electrophoresis. Moreover, it was confirmed thatthe protein has an amino acid sequence of GRP as determined by a peptidesequencer.

Example 2

Production of polyclonal antibody

The product DROGRP(31-98) thus prepared was conjugated with KLH ascarrier protein, and 150 μg of the conjugate was first injected to arabbit NZW, and a further 100 μg injected after two weeks, 100 μg after2 weeks, 85 μg after 6 days, 80 μg after 12 days and 60 μg after 12 dayswere injected. After an increase of antibody titer was confirmed, thewhole blood was obtained.

Example 3

Production of monoclonal antibody

DROGRP(31-98) was conjugated with KLH as carrier protein, and theconjugate was first injected to a Balb/c mouse, and a further injected50 μg after 3 weeks, 50 μg after three weeks, 50 μg after 4 weeks and 30μg after 4 weeks were injected.

Spleen cells (1×10⁸ cells/ml) from the immunized mouse were mixed withpreviously cultured myeloma cells P3U1 (1×10⁷ cells/ml) at a mixingratio of 10:1 by cell amount, and the mixture was incubated at 37° C.for 5 minutes.

Next, to the mixture was added 50% polyethylene glycol 1500 followed byRPMI 1640 medium. After centrifugation, HAT medium containing 20% FCSwas added thereon, and after adjusting cell concentration to 10⁵cells/ml, the cell suspension was distributed to wells of a 96 wellmicrotiter plate at a volume of 100 μl/well. Next, culturing was carriedout at 37° C. for 10 to 14 days.

To an ELISA plate coated with 100 ng/ml DROGRP(31-98) was added 100 μlof the above-obtained culture supernatant, which was reacted for 2hours. After reaction with peroxidase-labeled anti-mouse immunoglobulinG antibody for 1.5 hours, the reaction mixture was developed with a 33',55'-tetramethylbenzidine (TMBZ) solution. Absorbance at 450 nm wasmeasured, and clones providing at least 0.3 of the absorbance was takenas positive to obtain hybridoma that produces a desired monoclonalantibody. The hybridoma cells were intraperitoneally inoculated intomice treated with pristan, and monoclonal antibody produced in theascites was recovered. Alternatively, the hybridoma cells were culturedin vitro in a medium, and monoclonal antibody was obtained from theculture supernatant.

The monoclonal antibody was purified according to a conventionalprocedure by ammonium sulfate precipitation, dialysis to phosphatebuffer and purification by a protein A- linked SEPHARASE column toobtain an IgG fraction.

An affinity of the monoclonal antibody thus prepared to DROGRP(31-98)antigen was determined as follows. The DROGRP(31-98) antigen wasimmobilized to a microtiter plate to saturation, and to the plate wasadded 20 μl of a solution containing 0 to 0.3 μg/ml monoclonal antibodyto the DROGRP(31-98) prepared by dilution of the monoclonal antibody (10μg/ml), and allowed to react. The antibody bonded to the immobilizedDROGRP(31-98) was measured using an enzyme-labeled anti-mouse IgG.

An amount of the bonded antibody [B] was obtained from the relationshipbetween the values of absorbance measured and amounts of the antibodyadded, and an amount of free antibody (F) was obtained by subtracting anamount of the bonded antibody from an amount of the antibody added. Theamount of the bonded antibody [B] was converted to a molar concentrationassuming molecular weight of the antibody being 150,000 daltons, and tothe obtained molar concentrations the values of B/F wereScatchard-platted. An association constant Ka=3×10⁹ ˜2×10¹⁰ /M wasobtained from the slope of Scatchard plotting. From this value thedissociation constant Kd=3×10⁻⁸ ˜5×10⁻⁷ M was obtained using theequation Kd≈1/Ka.

Example 4

Assay of blood GRP (FIG. 3)

An ELISA plate was coated with 25 μg/ml monoclonal antibody toDROGRP(31-98) according to a conventional procedure, and after addingthereon 200 μl of DROGRP(31-98) standards or 200 μl of sera frompatients with small cell lung cancer, 200 μl of peroxidase-labeledpolyclonal antibody to DROGRP(31-98) was added to allow reaction. Afterwashing the ELISA plate, a TMBZ solution was added thereon to developthe color, absorbance at 450 nm was measured to prepare a calibrationcurve, and a concentration of GRP precursor in the blood was obtainedusing the calibration curve.

Example 5

Comparison of stability of GRPs in the blood (FIGS. 4A and 4B)

Stabilities in the blood of GRP(1-27) and DROGRP(31-98) were comparedusing antibodies to the above-prepared DROGRP(31-98) and antibodies tothe conventional GRP(1-27)

A small amount of GRP(1-27) or DROGRP(31-98) was added to a serum,plasma, or aprotinin--containing plasma, and residual concentration ofGRP(1-27) and DROGRP(31-98) were determined at 0, 1, and 6 hours fromthe addition, and percentages of the residual concentration wereobtained taking the concentration at the starting point (0 hour) as100%. As seen from FIG. 4, although the active peptide GRP(1-27) wasdegraded and disappeared in the presence of serum or plasma, the peptideDROGRP(31-98) was stable for a long time in the serum or plasma.

Example 6

Preparation of monoclonal antibody

The GRP(31-98) prepared as described above was conjugated to a carrierprotein, thyroglobulin, the conjugate was dissolved in phosphate buffer(pH 7.4)(PBS(-)) to a concentration of 1.0 mg/ml, and the solution wasmixed with a same volume of Freund's complete adjuvant to form asuspension. An amount of the suspension thus prepared containing 0.01 to0.05 mg of GRP(31-98) was intraperitoneally administered to a BALB/Cmouse of 4 to 6 weeks old. After about 12 weeks, the immunized animalwas administered with the same concentration of GRP(31-98) solution inPBS(-1) containing about 0.01 to 0.03 mg of DROGRP(31-98) through a tailvenous. After 3 days from the administration, the spleen was asepticallyremoved from the immunized animal.

Next, the spleen was disrupted to single cells by a mesh, and the cellswere washed three-times with RPMI-1640 medium. Mouse myeloma cells p3×63Ag 8 (Nature, 256, 495-497, 1975) in the logarithmic growth phase werecultured for a few days in the presence of 8-azaguanine to completelyeliminate reverse mutant, and washed as described above. 1.1×10⁷ cellsof the mouse myeloma cell line and 1.4×10⁸ spleen cells prepared asdescribed above were mixed. After centrifugation at 200_×g for 5minutes, the supernatant was replaced with 1 ml of RPMI-1640 mediumcontaining 50% PEG 4000 (Merck) warmed at 37° C. for cell fusion.

The cells subjected to the cell fusion were centrifuged to remove PEG,and cultured in RPMI-1640 medium containing hypoxanthine, aminopurineand thymidine (abbreviated as HAT hereinafter) as well as 15% fetal calfserum (FCS) for 1 to 2 weeks to allow for hybridomas exclusively togrow. Next, the hybridoma cells were grown in a HAT-free medium forabout two weeks, and clones were screened by ELISA as described below toobtain hybridoma producing a monoclonal antibody of the presentinvention showing a desired reaction specificity. The ELISA was carriedout as follows. DROGRP(31-98) was dissolved in phosphate buffer (pH7.4)(PBS) to a concentration of 1 μg/ml, and 50 μl each of the solutionwas distributed to each well of a 96-well microtiter plate, and theplate was incubated overnight at 4° C. or for one hour at roomtemperature for adsorption. After the adsorption, the wells were washedthree-times with 0.05% TWEEN 20-containing PBS(-) (abbreviated asT-PBS), and 200 μl/well of 1% BSA-containing PBS(-) was distributed andthe plate was incubated at room temperature for one hour. The 1%BSA-containing PBS(-) was removed, and 50 μl/well of hybridoma culturesupernatant, crude monoclonal antibody or purified monoclonal antibodywas added, followed by reaction for one hour at room temperature. Afterthe reaction, wells were washed three-times with T-PBS, and 50 μl/wellof an enzyme-labeled mouse IgG+M antibody (Jackson) diluted 5000-foldwith PBS(-) containing 1% BSA, 1% polyvinylpyrrolidone and 0.05% TWEEN20was added, and reaction was carried out at room temperature for 30minutes. Non-reacted antibody was eliminated by 4 washings with T-PBS,and 50 μl/well of orthophenylenediamine (OPD) solution (Wako PureChemicals) was added for reaction. After 20 minutes of incubation atroom temperature, the reaction was terminated with 2N sulfulic acid, andabsorbance at 492 nm was measured.

The hybridoma thus obtained were designated as proGRP-1E2, proGRP-2B10,proGRP-20D2, pro-GRP-3H1, proGRP-3G2 and proGRP-4C9, and among them thehybridoma proGRP-2B10 and proGRP-3G2 were deposited with FermentationResearch Institute, Agency of Industrial Science and Technology (FRI)under the Budapest Treaty on Dec. 9, 1992 as FERM BP-4110 and FERMBP-4109 respectively. Moreover, the hybridoma proGRP-20D2 was designatedas anti-proGRP monoclonal antibody-producing hybridoma (20D2) and wasdeposited with FRI under the Budapest Treaty on Feb. 10, 1993 as FERMBP-4184.

According to double immunodiffusion using rabbit anti-mouse Ig isotypeantibodies (Zymed), isotype of monoclonal antibodies produced by theabove-mentioned hybridoma was determined as follows. The monoclonalantibodies GRP-1E2, GRP-2B10 and GRP-3G2 produced by the hybridomaproGRP-1E2, proGRP-2B10 and proGRP-3G2 respectively were IgG1; themonoclonal antibody GRP-20D2 produced by the hybridoma proGRP-20D2 wasIgG2; and the monoclonal antibody GRP-3H1 produced by the hybridomaproGRP-3H1 was IgM.

Example 7

ELISA using monoclonal antibodies

The hybridoma proGRP-2B10, proGRP-1E2, proGRP-20D2, proGRP-3H1 andproGRP-409 were intraperitoneally inoculated in mice, and from theascites, corresponding monoclonal antibodies GRP-2B10, GRP-1E2,GRP-20D2, GRP-3H1 and GRP-409 with at least 90% purity were obtainedusing a Protein-A column, gel-filtration column or Protein-G column.

ELISA was carried out as follows. GRP(31-98) was dissolved in phosphatebuffer (pH 7.4) (PBS) to a concentration of 1 μg/ml, and 50 μl each ofthe solution was distributed to each well of a 96-well microplate, andthe plate was incubated overnight at 4° C. or for one hour at roomtemperature for adsorption. After the adsorption, the wells were washedthree-times with 0.05% TWEEN 20-containing PBS(-) (abbreviated asT-PBS), and 200 μl/well of 1% BSA-containing PBS(-) was distributed andthe plate was incubated at room temperature for one hour. The 1%BSA-containing PBS(-) was removed, and 50 μl/well of hydridoma culturesupernatant, crude monoclonal antibody or purified monoclonal antibodywas added, followed by reaction for one hour at room temperature. Afterthe reaction, wells were washed tree-times with T-PBS, and 50 μl/well ofan enzyme-labeled mouse IgG+M antibody (Jackson) diluted 5000-fold withPBS(-) containing 1% BSA, 1% polyvinylpyrrolidone and 0.05% TWEEN 20 wasadded, and reaction was carried out at room temperature for 30 minutes.Non-reacted antibody was eliminated by 4 washings with T-PBS, and 50μl/well of orthophenylenediamine (OPD) solution (Wako Pure Chemicals)was added for reaction. After 20 minutes of incubation at a roomtemperature, the reaction was terminated with 2N sulfuric acid, andabsorbance at 492 nm was measured. The result is shown in FIG. 5. Asseen from FIG. 5, monoclonal antibody most highly reactive with proGRP(GRP precursor) is GRP-3G2, followed by GRP-2B10, GRP-1E2, GRP-20D2,GRP-3H1 and GRP-4C9 in this order.

Example 8

Production polyclonal antibody

The polypeptide proGRP(31-98) prepared as described above was conjugatedto a carrier protein, thyroglobulin, the conjugate was dissolved inphosphate buffer (pH 7.4) (PBS(-)) to a concentration of 1.0 mg/ml, andthe solution was mixed with a same volume of Freund's complete adjuvantto form a suspension. An amount of the suspension thus obtainedcontaining 0.1 mg of GRP(31-98) was subcutaneously administered to1.5-2.0 kg weight rabbit Kb1:JW of 8 weeks old, followed by 6 repeatedadministrations of 0.07 mg of proGRP(31-98) with ten days intervals. Theblood from rabbits which provided a high titer was obtained to preparepolyclonal antibody.

a sandwich ELISA was accomplished using a rabbit anti-proGRP polyclonalantibody as well as the above-mentioned monoclonal antibodies GRP-3G3and GRP-2B10, as follow.

The monoclonal antibodies GRP-3G2 and GRP-2B10 were diluted with PBS toconcentrations of 7 μ/ml and 7 μg/ml respectively (total 14 μg/ml) toform a solution, 100 μl of the diluted monoclonal antibody solution wasadded to each well to coat the wells at 4° C. overnight. The wells werewashed twice with PBS, after addition of 350 μl of 0.5% casein/PBS towells, incubated at room temperature for 2 hours, and washed twice withPBS. 50 μl/well of a sample-diluting solution (0.1m phosphate buffer pH7.1, containing 1% BSA, 1% PVP, 0.05% casein, 0.05% TWEEN 20, 10 mMEDTA, and 0.5M NaCl) was added, 50 μl of a sample was added, and afterreaction at 37° C. for 2 hours, the wells were washed 5-times with PBScontaining 0.05% TWEEN 20.

Next, 100 μl/well of peroxidase-labeled rabbit anti-proGRP antibodydiluted to 5 μg/ml with a labeled antibody diluting solution was addedand an incubation was carried out at room temperature for one hour.Next, the wells were washed 5-times with 0.5% TWEEN 20/PBS, 100 μl/wellof a substrate solution (orthophenylene diamine (OPD) solution) wasadded, and after a reaction at room temperature for 30 minutes, thereaction was terminated with 100 μl/well of 2N sulfuric acid. A resultobtained by varying concentrations of proGRP(31-98) is shown in FIG. 6.It it considered that the detection limit is about 3 pg/ml, and themeasuring range is 10 to 800 pg/ml

In addition, result obtained by using samples prepared by diluting aserum of a patient considered to have a high proGRP value with a serumof a normal person at different dilution ratios is shown in FIG. 6.Since a line obtained in the former experiment using a recombinantproGRP(31-98) and a line obtained in the latter experiment usingproGRP-containing serum are approximately parallel, it is believed thatthe reactivity of recombinant proGRP(31-98) with antibodies is similarto the reactivity of a serum proGRP with antibodies.

Next, 7 sera from healthy subjects and 5 sera from patients with smallcell lung cancer were tested, and the results are shown in the followingtable.

    ______________________________________                                        proGRP value in serum                                                                             Patient with                                              Healthy             small cell                                                subjects                                                                             proGRP pg/ml lung cancer proGRP pg/ml                                  ______________________________________                                        No. 1  20.0         No. 1       18,300                                        No. 2  39.0         No. 2       21,300                                        No. 3  5.7          No. 3         352                                         No. 4  7.4          No. 4         143                                         No. 5  3.8          No. 5        3,200                                        No. 6  8.7                                                                    No. 7  24.5                                                                   ______________________________________                                    

As seen from the above results, the highest value of proGRP of healthysubjects is 39 pg/ml, while the values of proGRP of patients with smallcell lung cancer are at least 143 pg/ml, the highest value in thepatients is 21,300 pg/ml. There is a big difference between proGRP valuein serum from a healthy subject and that from a patient with small celllung cancer, demonstrating that proGRP is highly useful as a marker fordiagnosing small cell lung cancer.

In summary, since the present antibody to an inactive region in GRPprecursor, i.e., proGRP(31-98) has a high affinity to GRP precursor andGRP precursor is highly stable in the blood in comparison with activeGRP, then lung cancer, especially small cell lung cancer, can bediagnosed with high reliability by detecting or measuring GRP precursorin the blood using the present antibodies.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 204 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..204                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGTACTGGTGAGAGCTCTTCTGTTTCTGAACGTGGATCCCTTAAGCAG48                            SerThrGlyGluSerSerSerValSerGluArgGlySerLeuLysGln                              151015                                                                        CAGCTTCGCGAATACATCCGTTGGGAAGAAGCTGCTCGTAACCTGCTA96                            GlnLeuArgGluTyrIleArgTrpGluGluAlaAlaArgAsnLeuLeu                              202530                                                                        GGCCTGATCGAAGCTAAAGAAAACCGTAACCACCAGCCGCCGCAGCCG144                           GlyLeuIleGluAlaLysGluAsnArgAsnHisGlnProProGlnPro                              354045                                                                        AAAGCTTTAGGTAACCAGCAGCCGTCTTGGGACTCTGAAGACTCTTCG192                           LysAlaLeuGlyAsnGlnGlnProSerTrpAspSerGluAspSerSer                              505560                                                                        AACTTTAAAGAC204                                                               AsnPheLysAsp                                                                  65                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       SerThrGlyGluSerSerSerValSerGluArgGlySerLeuLysGln                              151015                                                                        GlnLeuArgGluTyrIleArgTrpGluGluAlaAlaArgAsnLeuLeu                              202530                                                                        GlyLeuIleGluAlaLysGluAsnArgAsnHisGlnProProGlnPro                              354045                                                                        LysAlaLeuGlyAsnGlnGlnProSerTrpAspSerGluAspSerSer                              505560                                                                        AsnPheLysAsp                                                                  65                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AATTCATGAGTACTGGTGAGAGCTCTTCTGTTTCTGAACGTGGATCC47                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CTTAAGCAGCAGCTTCGCGAATACATCCGTTGGGAAGAAGCTGCTCGTAACCTG54                      (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTAGGCCTGATCGAAGCTAAAGAAAACCGTAACCACCAGCCGCCGCAGCCGAAA54                      (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCTTTAGGTAACCAGCAGCCGTCTTGGGACTCTGAAGACTCTTCGAACTTTAAAGACTAA60                TAAG64                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CTTAAGGGATCCACGTTCAGAAACAGAAGAGCTCTCACCAGTACTCATG49                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GCCTAGCAGGTTACGAGCAGCTTCTTCCCAACGGATGTATTCGCGAAGCTGCTG54                      (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TAAAGCTTTCGGCTGCGGCGGCTGGTGGTTACGGTTTTCTTTAGCTTCGATCAG54                      (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TCGACTTATTAGTCTTTAAAGTTCGAAGAGTCTTCAGAGTCCCAAGACGGCTGCTGGTTA60                CC62                                                                          __________________________________________________________________________

We claim:
 1. A monoclonal antibody having an association constant K_(a)of 3×10⁹ -2×10¹⁰ /M obtained from a hybridoma cell line selected fromthe group consisting of proGRP-2B10 (FERM BP-4110), proGRP-3G2 (FERMBP-4109), and proGRP-2OD2 (FERM BP-4184).
 2. A hybridoma cell lineselected from the group consisting of proGRP-2B10 (FERM BP-4110),proGRP-3G2 (FERM BP-4109), and proGRP-2OD2 (FERM BP-4184).
 3. Adiagnostic agent for lung cancer comprising a monoclonal antibodyaccording to claim 1 and a carrier.